A previously undocumented transition in embryonic gene activity has been identified in the amphibian, Xenopus. Genes transcribed by RNA polymerase III achieve a somatic pattern of expression by repressing selective templates during a gastrula-neurala transition (GNT). Our preliminary studies show that the GNT is complete by the onset of neurulation (60,000 cells) and is independent of transcription factor concentration. The proposed research program will be directed toward determining the mechanism responsible for this extensive and permanent repression of the previously active genes. Experiments will make use of an in RNA polymerase III chromatin transcription assay and will address the relationship between the changing embryonic cell cycle and regulation of gene activity. Embryonic chromatin will be isolated at regular intervals to determine the exact timing of the GNT. The temporal order of gene-specific replication will be.examined with respect to the decline in transcriptional activity during the transition period. The cause and effect relationship between replication timing and gene expression will be addressed by increasing the number of transcriptionally active oocyte 5S RNA genes via microinjection of the MRNA for the 5S RNA gene-specific transcription factor TFIIIA. We can then determine whether replication timing of these normally inactive genes is altered by placing these sequences in an active conformation. The potential role of histone HI in controlling gene expression during the GNT will be tested by microinjection of synthetic HI MRNA to effectively increase the concentration of the HI protein in vivo. Finally, an in vitro assay system which reproduces the entire progression of regulated gene activity will be developed. This will permit a subtractive analysis by immunodepletion along with other reductionist approaches.